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CURRENT RESEARCH

1. The role of IL-23 in bone destruction

2. Developing novel inhibitors for the treatment of autoimmune diseases

3. The IL-23/IL-17 axis in psoriatic arthritis

4. Involvement of leukotriene B4 in Juvenile Arthritis (JA)

5. Autophagy in skeletal development


1. The role of IL-23 in bone destruction

We have previously shown that IL-23 plays a critical role in osteoclast differentiation and activation. Specifically, IL-23 in vivo gene transfer induces myelopoiesis and osteoclast differentiation in a Th17 independent manner. We are translating this data using human osteoclasts isolated from peripheral blood mononuclear cells (PBMC’s), with ongoing efforts to further define the cellular and molecular events that orchestrate the responses elicited by IL-23.

Systemic IL-23 induces joint inflammation and extensive bone loss in vivo

Figure 1. Systemic IL-23 induces joint inflammation and extensive bone loss in vivo. a) H&E-stained sections of the knee joint showing extensive bone loss, hyperplastic bone marrow, and pannus formation 30 days post IL-23 MC injection compared to GFP MC control (bars represent 500mm (top) and 250mm (bottom). b) Clinical disease severity score c) 3D rendered images from the paw and anterior and side knee views 60 days post IL-23 MC injection. d) Day 90 whole body scan of control mice compared to the IL-23 MC injected mice showing severe loss of bone mineral density in IL-23 MC injected mice.



2. Developing novel inhibitors for the treatment of Autoimmune diseases

We have partnered with Tanabe Research Laboratories, USA, a biotech company based in La Jolla, San Diego, California to develop novel inhibitors for the treatment of autoimmune disorders. TRL and Anaphore will generate novel trivalent proteins called Atrimers that can be programmed to bind to and activate or inhibit targets of interest. The partnership will focus on strategies to develop biologic therapies for autoimmune disorders such as, rheumatoid arthritis, inflammatory bowel disease, and psoriasis.

trivalent structure of Atrimer drug candidates affords three binding regions

Figure 2. PBMC and CD14+ cells cultured on coverslips (Left) for 18 days with MCSF and IL-23 showing Phalloidin, DAPI staining, and merged image of F-actin ring formation (Bars 25μm). The trivalent structure of Atrimer drug candidates (right) affords three binding regions, each containing loops of amino-acid sequences that can be programmed to yield a drug candidate that binds to its target with high potency and exquisite specificity.



3. The IL-23/IL-17 axis in psoriatic arthritis

Psoriatic arthritis (PsA) is an autoimmune disease where the interaction of the immune and skeletal system often results in bone loss. We have previously shown that IL-17A upregulates the receptor RANK on human osteoclast precursors to increase their responsiveness to RANKL leading to increased bone loss in vitro. In this project, we are investigating the cellular and molecular interactions that lead to epidermal hyperplasia and arthritis as it's commonly seen in psoriatic arthritis. We developed recombinant mini-circle DNA carrying the IL-23, IL-17A and RANKL genes and evaluate the effects of mini-circle-mediated gene transfer in vivo.
research figure3
Figure 3.  IL-17A exacerbates synovial inflammation and bone loss in inflammatory arthritis.  a) In vivo whole body and b) ex vivo 2D fluorescence imaging of C57BL/6 mouse liver explants one day post-gene transfer of IL-17A or GFP. c) Representative photographic images of inflamed mouse paws post-GFP (left) and post-IL-17A (right) gene transfer at day 37 post collagen-induced arthritis initiation showing severe inflammation in the IL-17A gene transfer. d) H&E staining of tissue sections of paws showing infiltration of mononuclear cells, synovial lining cell hyperplasia, destruction of joint cartilage layers, and fibrous ankylosis post IL-17A gene transfer. e) Micro-CT of mouse paws showing severe bone erosion present in IL-17A MC compared to GFP controls. 


4. Involvement of leukotriene B4 in Juvenile Arthritis (JA)

A critical issue in JA development that remains unresolved is how autoimmune processes are linked to a localized onset of inflammation in the joints. LTB4 is a potent proinflammatory mediator that causes inflammatory cell infiltration and also has direct effects on osteoclast differentiation and activation. The temporal correlation between the LTB4 generation and the onset of arthritis phenotype suggests that LTB4 may represent a critical link between autoimmune processes and the onset of joint inflammation. Therefore, targeting LTB4 synthetic or signaling pathways may represent a novel and effective therapeutic strategy for the treatment of JA.

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Figure 4.  Leukotriene biosynthetic pathways and receptors. The biosynthesis of leukotrienes can be triggered by a variety of stimuli with subsequent increase of intracellular calcium level. This actives cPLA2, which catalyzes the release of AA from membrane phospholipids. Subsequently activated 5-LO, together with its activating protein FLAP, converts free arachidonic acid into the unstable intermediate LTA4, LTA4 is then either hydrolyzed by LTA4 hydrolase to form LTB4, or conjugated with glutathione by LTC4S to form LTC4, LTC4 is further metabolized to LTD4 and LTE4. LTC4, LTD4 and LTE4 are collectively referred to as cystineyl leukotrienes. Once out of the cell, both LTB4 and cysteinyl leukotrienes act by binding to their specific receptors. There are two receptors for LTB4 named as BLT1 and BLT2, and 2 receptors for cysteinyl leukotriene named as cysLT1 and cysLT2. LTB4 is a potent chemoattractant for polymorphoneuclear leukocyte, monocyte macrophage and T cell, it also activates functional responses in these cells, including degranulation, reactive oxygen species generation, phagocytosis and cytokine production. The cysteinyl leukotrienes cause brochoconstriction, enhanced vascular permeability and mucus secretion.



5. Autophagy in skeletal development

Autophagy is a process by which the cell degrades its own components by first sequestering them to specialized vacuolar organelles, called autophagosomes and then fusing these vacuoles with lysosomes. Autophagy proteins have been shown to regulate the secretory component of osteoclastic bone resorption and TNF-mediated joint destruction in experimental arthritis. Therefore the implications of autophagy in the pathogenesis of inflammatory arthritis may present us with new treatment avenues in combating inflammatory musculoskeletal diseases.

research figure5Figure 5. Autophagy in osteoclasts: A) Selective macroautophagy and bone degradation utilize lysosomal machinery to perform their biological processes. B) Autophagsomal machinery is also involved in transcytosis of bone debris containing vesicles. We have identified protein domains associated with vesicle trafficking that play crucial roles in both autophagy and bone destruction. (Faint and bold black arrows indicate macroautophagy and bone debris containing vesicles respectively. Yellow arrows indicate the multiple trancytosis routes).